In stimulated emission depletion (STED) microscopy, the image resolution can be achieved well below the diffraction limit of conventional microscopy (see for instance FIG. 2 in B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle and S. W. Hell, “Resolution scaling in STED microscopy”, Optics Express 16 (2008) p. 4154-4162. In STED microscopy, a fluorescence marker is excited by a pulsed laser system. Shortly after this excitation i.e. within the decay time of the fluorescence a second laser pulse is applied having a wavelength close to the wavelength of the emission wavelength of the fluorescence. This second laser pulse has a donut shaped spot (or other similar shapes differing from the shape of the first pulse). Due to stimulated emission this second pulse will de-excite the fluorescence molecules within the donut shaped spot region. However the molecules within the center of the donut will not be de-excited. This center region is in general smaller than the diffraction limited spot size of the conventional microscope. Hence detecting the fluorescence coming from the remaining excited molecules after the two laser pulses originate from a region smaller than the diffraction limit, hence producing sub diffraction limited resolution of imaging. This has recently attracted some attention in this technical field.
To produce the two pulsed laser beams typically the optical setup shown in FIG. 1 of the above reference by Harke et al. is used. This causes severe constraints on the beam alignment in all three spatial dimensions, and typically re-alignment is periodically needed due to mechanical, thermal, and/or optical beam drift in the optical setup. Furthermore, the depletion beam requires different optical components in order to produce the donut shape spot. Similarly, the two laser beams puts a constraint on the possible down-scaling of the optical STED microscopy, which is of paramount importance for in-vivo medical and biological imaging.
In connection with an optical fiber scanner, cf. for example international patent application WO 2009087527 by the present applicant, it may be mentioned that for such applications one generally prefers to have a common optical beam path for the excitation beam and the STED beam.
The inventors of the present invention have appreciated that an improved STED microscopy system is of benefit, and has in consequence devised the present invention.